Abstract
Reports have shown that a certain level of reactive oxygen species (ROS) can promote mitochondrial DNA (mtDNA) replication. However, it is unclear whether it is the mitochondrial ROS that stimulate mtDNA replication and this requires further investigation. Here we employed a photodynamic system to achieve controlled mitochondrial singlet oxygen (1O2) generation. HeLa cells incubated with 5-aminolevulinic acid (ALA) were exposed to laser irradiation to induce 1O2 generation within mitochondria. Increased mtDNA copy number was detected after low doses of 630 nm laser light in ALA-treated cells. The stimulated mtDNA replication was directly linked to mitochondrial 1O2 generation, as verified using specific ROS scavengers. The stimulated mtDNA replication was regulated by mitochondrial transcription factor A (TFAM) and mtDNA polymerase γ. MtDNA control region modifications were induced by 1O2 generation in mitochondria. A marked increase in 8-Oxoguanine (8-oxoG) level was detected in ALA-treated cells after irradiation. HeLa cell growth stimulation and G1-S cell cycle transition were also observed after laser irradiation in ALA-treated cells. These cellular responses could be due to a second wave of ROS generation detected in mitochondria. In summary, we describe a controllable method of inducing mtDNA replication in vitro.
Highlights
We investigated the kinetics of protoporphyrin IX (PpIX) accumulation in HeLa cells following incubation with 200 uM Aminolevulinic acid (ALA) for up to 12 h by fluorescence measurement at 630 nm
The addition of 10 μ M Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 4 h stopped the accumulation of PpIX, while removal of CCCP at 8 h allowed the gradual recovery of PpIX incorporation in the following hours (Fig. 1A)
In photodynamic therapy (PDT), particular attention has been focused on the ability of 1O2 to trigger cell death, and it is unclear whether 1O2 can act as a signaling agent to regulate certain biological events, such as mitochondrial biogenesis
Summary
Endogenous or added for this purpose. This process provides an approach for the controlled production of 1O2 within mitochondria. 5-Aminolevulinic acid (ALA) is the common precursor of tetrapyrrole compounds, and is widely distributed in both plant and animal cells[8,9]. The generation of 1O2 within mitochondria, induced by laser irradiation, allowed us to determine whether mitochondrial ROS, such as 1O2, can act as a signaling molecule to facilitate mtDNA replication. A temporary mtDNA copy increase within 4 h after laser irradiation was seen in ALA-treated cells (Fig. 2B).
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